Light-Activatable TET-Dioxygenases Reveal Dynamics of 5-Methylcytosine Oxidation and Transcriptome Reorganization

Abstract

Ten-eleven-translocation (TET) dioxygenases catalyze the oxidation of 5-methylcytosine (5mC), the central epigenetic regulator of mammalian DNA. This activity dynamically reshapes the epigenome and transcriptome by depositing oxidized 5mC derivatives and initiating active DNA demethylation. However, studying this dynamic is hampered by the inability to selectively activate individual TETs with temporal control in cells. We report activation of TETs in mammalian cells by incorporation of genetically encoded 4,5-dimethoxy-2-nitrobenzyl-L-serine as a transient active-site block, and its subsequent deprotection with light. Our approach enables precise insights into the impact of cancer-associated TET2 mutations on the kinetics of TET2 catalysis in vivo, and allows time-resolved monitoring of target gene activation and transcriptome reorganization. This sets a basis for dissecting the order and kinetics of chromatin-associated events triggered by TET catalysis, ranging from DNA demethylation to chromatin and transcription regulation.

Catalytic Space Engineering as a Strategy to Activate C–H Oxidation on 5-Methylcytosine in Mammalian Genome

“…Ascorbic acid, however, activates several 2KG-dependent dioxygenases, obscuring its exclusive role in controlling TET activity. Other approaches focus on conditional reconstitution of wild-type TET2, which are less likely to regulate the 5mC ox distribution. , …”

“…Other approaches focus on conditional reconstitution of wild-type TET2, which are less likely to regulate the 5mC ox distribution. 11,12 We envisioned an active site remodeling tactic to stimulate C−H oxidation for enriching the human genome with the lowabundant intermediates for functional analysis. In TETmediated C−H oxidation, hydrogen abstraction by oxo-ferryl species is the rate-limiting step with a relative activation barrier following the order 5fC > 5hmC > 5mC (Figure 1B).…”

Advances and Opportunities in Epigenetic Chemical Biology

“…This approach was used to interrogate the kinetics of TET enzymes, allowing the direct observation of 5hmC formation upon TET activation. [60] Similarly, another group reported a photo-activatable isocitrate dehydrogenase (IDH) using a photo-caged lysine. This enabled the study of an IDH2 variant found in cancers, which generates (R)-2-hydroxyglutarate (2HG) instead of alpha-ketoglutarate.…”

“…For example, a recent study used genetic code expansion ((GCE), see below) to incorporate a photocaged serine into TET enzymes, allowing them to be activated with light in mammalian cells. This approach was used to interrogate the kinetics of TET enzymes, allowing the direct observation of 5hmC formation upon TET activation [60] . Similarly, another group reported a photo‐activatable isocitrate dehydrogenase (IDH) using a photo‐caged lysine.…”

Integrative 5-Methylcytosine Modification Immunologically Reprograms Tumor Microenvironment Characterizations and Phenotypes of Clear Cell Renal Cell Carcinoma

“…The m 5 C modification was the first discovered epigenetic marker and plays an important role in regulating the transcriptome profiles and carcinogenesis process of solid tumors, which often harbor aberrant DNA methylation ( Martisova et al, 2021 ). The m 5 C modification is frequently found in large clusters called CpG islands, which are present in gene-promoter regions and suppress gene transcription ( Chen et al, 2019 ; Palei et al, 2020 ). A series of enzymes, called writers, readers, and erasers, is responsible for adding, recognizing, and removing the m 5 C modifications, respectively ( Rausch et al, 2020 ).…”